Amino acid compositions and use thereof in immunosuppression

ABSTRACT

The present invention provides for the use glycine in the preparation of a medicament or nutritional formulation for the prophylaxis and/or therapy of renal dysfunction induced by cyclosporins or ascomycins.

This Application is a continuation of U.S. Ser. No. 08/690,476, filedJul. 30, 1996, now pending.

The present invention relates to the use of glycine in the preparationof a medicament or nutritional formulation which may be therapeuticallyadministered to patients to prophylactically and/or therapeuticallytreat renal dysfunction induced by substances belonging to the classesof cyclosporins and/or ascomycins.

Cyclosporins comprise a class of structurally distinct, cyclic,polyN-methylated undecapeptides, generally possessing immunosuppressive,anti-inflammatory, anti-viral and/or anti-parasitic activity, each to agreater or lesser degree. The first of the cyclosporins to be identifiedwas the fungal metabolite Cyclosporin A, or Ciclosporin, and itsstructure is given in The Merck Index, 11th Edition; Merck & Co., Inc.;Rahway, N.J., U.S.A. (1989) under listing 2759. Later cyclosporins to beidentified are cyclosporins B, C, D and G which are also listed in TheMerck Index under listing 2759. A large number of synthetic analoguesare also known and representative examples are disclosed in EP 296 123,EP 484 281, and GB 2222770. Cyclosporin A and its structurally similaranalogues and derivatives as well as metabolites thereof are generallyreferred to as “cyclosporins” for the purposes of this specification.

Ascomycins, of which FK-506 is the best known, are another class ofgenerally immunosuppressive substances, also referred to as macrolideimmunosuppressants. FK-506 is a macrolide immunosuppressant that isproduced by Streptomyces tsukubaensis No. 9993. The structure of FK-506is given in the appendix to The Merck Index, supra, as item A5. A largenumber of related compounds which retain the basic structure andimmunological properties of FK-506 are also known. These compounds aredescribed in various publications, for example EP 184162, EP 315973, EP323042, EP 423714, EP 427680, EP 465426, EP 474126, WO 91/13889, WO91/19495, EP 484935, EP 532088, EP 532089, WO 93/5059 and the like.Ascomycin, FK-506 and their structurally similar analogues andderivatives as well as metabolites thereof are termed collectively“ascomycins” in this specification.

Due to their extremely useful pharmaceutical properties, cyclosporins(Cyclosporins A and G in particular) and ascomycins (e.g., FK-506) havewide application in, for example the prevention of transplant rejection,in the treatment of auto-immune diseases such as rheumatoid arthritisand psoriasis, and also in the treatment of multi-drug-resistance.Cyclosporins and ascomycins also have certain side effects, the mostnotable being renal dysfunction, in particular nephrotoxicity,especially at higher doses. Nephrotoxicity is characterized bydiminished renal blood flow and glomerular filtration with correspondingelevations in serum creatinine, alkaline phosphatase and urea as well asproximal cell swelling and necrosis and infiltration of macrophages. Insome studies, hypoxia has been shown to damage proximal tubules ratherselectively, and a decrease in blood flow could lead to hypoxia.Evidence has been presented implicating intracellular calcium in thispathology, and calcium channel blockers are effective in minimizinginjury. However, Ca⁺⁺ channel blockers cannot be given without impunity.Accordingly, there is currently no known useful therapy for thisimportant side effect on the kidney.

It has now surprisingly been found that glycine is suitable forprophylactic and/or therapeutic treatment of renal dysfunction inducedby cyclosporins or ascomycins.

In accordance with the invention it has more particularly been foundthat dietary glycine inhibits and/or ameliorates one of the major sideeffects of chronic cyclosporin A administration-nephrotoxicity. Glycineprevents the decrease in glomerular filtration rate. Glycine alsoprevents the elevation in serum creatinine and urea, and tends tominimize the elevation in alkaline phosphatase due to cyclosporin A. Theswelling and necrosis of proximal tubules and macrophage infiltrationare also all prevented by dietary glycine. Thus, it is clear thatglycine has a major protective action on nephrotoxicity due tocyclosporin A.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting the mean serum creatinine levels (mg/dl)observed in four treatment groups of rats.

FIG. 2 is a bar graph depicting the mean glormerular filtration rateobserved in each of the treatment groups following four weeks oftreatment.

FIG. 3A is a bar graph depicting the mean number of dilated proximaltabules per slide observed in each of the treatment groups under lowpower (100×).

FIG. 3B is a bar graph depicting the mean number of necrotic cells ineach of the treatment groups counted on five different tubules per slideunder high power (400×).

FIG. 4 is a bar graph depicting the mean number of macrophages observedproximate to 5 different tubules per slide under high power (400×).

FIG. 5 is a bar graph depicting the percentage of neutrophils found as afunction of the total number of white blood cells in the control and CsAtreatment groups.

In view of the above-mentioned effects, there are medicaments and/ornutritional formulations comprising glycine as well as methods of usingglycine. For use in the medicaments, nutritional formulations andmethods of the invention, glycine is conveniently employed in free aminoacid form (as opposed to salt form), in the form of glycine precursors,in particular alanine or serine (L-alanine, L-serine), likewise in freeamino acid form, in physiologically acceptable salt form of said aminoacids, or in form of mixtures of said amino acids and/or physiologicallyacceptable salts thereof. Glycine is preferably used in free amino acidform (as opposed to salt form), in physiologically acceptable salt formor in the form of a mixture of glycine in free amino acid form withglycine in physiologically acceptable salt form; most preferably glycineis in free amino acid form. Glycine may also be used in the form ofdipeptides according to the invention.

This invention therefore provides the use of glycine in the preparationof a medicament or nutritional formulation for the prophylaxis and/ortherapy of renal dysfunction induced by cyclosporins or ascomycins.

The invention further provides a method for the prophylactic and/ortherapeutic treatment of renal dysfunction induced by cyclosporins orascomycins comprising administering to a human being or other mammalprior to, simultaneous with, or subsequent to the treatment with acyclosporin or ascomycin a medicament or nutritional formulationcomprising glycine in an amount effective to inhibit and/or amelioratethe renal dysfunction.

Still further the invention provides a medicament or nutritionalformulation comprising glycine for the prophylaxis and/or therapy ofrenal dysfunction induced by cyclosporins or ascomycins.

The invention also provides a medicament or nutritional formulationcomprising a therapeutically effective amount of a cyclosporin orascomycin in combination with glycine in an amount effective to inhibitand/or ameliorate the renal dysfunction induced by the cyclosporin orascomycin.

Glycine is particularly useful in the prophylactic and/or therapeutictreatment of the renal dysfunction nephrotoxicity.

Glycine is further particularly useful for the therapy and/orprophylaxis of renal dysfunction induced by the cyclosporins cyclosporinA (Ciclosporin) and PSC 833([3′-desoxy-3′-oxo-MeBmt]¹[Val]²-Ciclosporin, e.g. as described in EP296 122), particularly by cyclosporin A.

Glycine is also particularly useful for the therapy and/or prophylaxisof renal dysfunction induced by the ascomycins FK-506 and ASM(33-epi-chloro-33-desoxy-ascomycin, e.g. as described in Ex. 66a of EP427 680), particulary by FK-506.

Furthermore, glycine is particularly useful for the prophylaxis and/ortherapy of renal dysfunction induced by immunosuppressive cyclosporinsor ascomycins.

The nutritional formulation or medicanent may be administered to thepatient enterally or parenterally. The enteral administration route ispreferred. Particularly contemplated enteral administration routes areoral administration and/or tube feeding. The medicament or formulationis conveniently administered in the form of an aqueous liquid. Themedicament or formulation in a form suitable for enteral application isaccordingly preferably aqueous or in powder form, whereby the powder isconveniently added to water prior to use. For use in tube feeding, theamount of water to be added will depend, inter alia, on the patient'sfluid requirements and condition. It will be appreciated that, for acutetreatment, the parenteral application route is preferred.

The medicament or formulation may be so formulated as to deliver to thepatient from 1 to 80 g, preferably 10 to 60 g, particularly preferred 15to 30 g of glycine per 24 hours. The amount of medicament or formulationto be administered depends to a large extent on the patients' specificrequirements. Such daily amounts of glycine are suitable for treatmentof the desired effects as well as for prophylactic/pretreatment. Theglycine comprising medicament or formulation may be administered to thepatient in an amount such that the concentration of glycine in thepatients' plasma is elevated to between 0.5 and 2.0 mM, preferably from1.0 to 2.0 mM. Whilst concentrations higher than this are anticipated,it is expected that significant clinical effects will be obtained if theconcentration of the acid is increased, as a consequence ofadministration of the formulation or medicament, so that it lies in therange of from 1.2 to 1.5 mM. In traumatic, hypercatabolic patients itmay even be beneficial to raise the plasma glycine, serine or alaninelevels to about 0.2 to 0.3 mM which corresponds to plasma glycine levelsof healthy individuals.

Preferably the medicament or nutritional formulation comprises onlyglycine and optionally L-arginine or L-ornithine as free amino acids (asopposed to protein-bound or peptide-bound amino acids).

When glycine is administered in the form of a medicament such amedicament will comprise from 0.1 to 99 g of glycine per 100 g.

In general, favourable effects are obtained when administering glycinein the form of a nutritional formulation, which may, depending on thecircumstances be a complete formula diet (i.e. a diet supplyingessentially all required energy, amino acids, vitamins, minerals andtrace elements) or a dietary supplement. A dietary supplement typicallyprovides for 5 to 20% of the total daily calory intake, preferably about10%. The nutritional formulation will conveniently be taken in aqueousliquid form. A nutritional formulation accordingly may comprise a sourceof carbohydrates, lipids fat (fat source) and/or protein (nitrogensource), and glycine, characterized in that glycine is present in theformula diet in an amount of about 0.2 to 60 g per 100 g dry weight offormula, preferably 5 to 45 g, particularly preferred 7.5 to 30 g per100 g dry weight. The nutritional formulation will preferably furthercomprise other nutritionally advantageous components such as vitamins,minerals, trace elements, fibers (preferably soluble fibers). Theamounts of carbohydrates lie in the range of 0-85% by weight, preferably25 to 75% by weight, the amount of fat in the range of 0-30% by weightand the amount of protein in the range of 0-75% by weight, preferably 15to 65%. Preferred nutritional formulations comprise the following: adietary supplement consisting essentially of glycine and carbohydrates,a dietary supplement consisting essentially of glycine, carbohydratesand protein and/or a complete formula diet consisting essentially ofglycine, carbohydrate, protein, fat, vitamins, minerals, trace elementsand optionally fibers. Such dietary supplements or complete formuladiets preferably further comprise L-arginine or other physiologicallyacceptable compound associated with the synthesis of nitric oxides suchas Lornithine or glycerol, whereby L-arginine is preferred.

Examples of suitable nitrogen sources include nutritionally acceptableproteins such as soy bean or milk-derived proteins such as whey,caseinates or skim milk powder, and/or protein hydrolysates thereof.Suitable carbohydrate sources include all sugars, maltodextrins andstarches. Examples of suitable fat sources include triglycerides, aswell as di- and monoglycerides.

Examples of vitamins suitable for incorporation into the medicament orformulation of the invention include Vitamin E, Vitamin A, Vitamin D,Vitamin K, folic acid, thiamin, riboflavin, Vitamin B₁, B₂, B₆ and B₁₂,niacin, biotin and panthotenic acid in nutritionally acceptable form.

Examples of mineral elements and trace elements suitable forincorporation into the medicament or formulation include sodium,potassium, calcium, phosphorous, magnesium, manganese, copper, zinc,iron, selenium, chromium, and molybdenum in nutritionally acceptableform.

In particular, the medicament or formulation will preferably comprisebeta-carotene (Vitamin A), Vitamin E, Vitamin C, thiamine, Vitamin B₁₂,choline, selenium and zinc in nutritionally acceptable form.

The term “soluble fiber” as used herein refers to fibers which are ableto undergo substantial fermentation in the colon ultimately to produceshort chain fatty acids. Examples of suitable soluble fibers includepectin, guar gum, locust bean gum, xanthan gum which may optionally behydrolysed. For adults the total amount of soluble fibre per day willconveniently lie in the range of from 3 to 30 g.

It may be indicated to use glycine in combination with one or more ofthe following components:

(i) omega-3 polyunsaturated fatty acids (PUFAs) where desired inadmixture with omega-6 PUFAs;

(ii) Larginine or other physiologically acceptable compound associatedwith the synthesis of nitric oxide, or mixtures thereof; and

(iii) a nucleobase source.

Whereby the use of a medicament or nutritional formulation comprisingglycine in combination with arginine or other physiologically acceptablecompound associated with the synthesis of polyarnines such as ornithineis preferred. Use of a medicament or nutritional formulation comprisingglycine, arginine or ornithine and omega-3 polyunsaturated fatty acids(PUFAs) is also preferred.

Nucleobase sources suitable for use in combination with glycine compriseor consist of natural nucleobases, nucleosides, nucleotides, RNA, DNA,equivalents thereof and/of mixtures comprising one or more of thesecompounds.

Natural nucleobases include the purines adenine and guanine as well asthe pyrimidines cytosine, thymine and uracil. Where the nucleobasesource is in the form of free nucleobases, it is preferably uracil.

Natural nucleosides include the ribose nucleosides adenosine, guanosine,uridine and cytidine and the deoxyribose nucleosides deoxyadenosine,deoxyguanosine, deoxythymidine and deoxycytidine.

Natural nucleotides include phosphate esters of natural nucleosides,such as the monophosphates adenylate (AMP), guanylate (GMP), uridylate(UMP), cytidylate (CMP), deoxythymidiylate (dTMP), deoxycytidylate(dCMP), and diphosphates and triphosphates of natural nucleosides suchas ADP and ATP.

A purified nucleobase source, such as yeast is preferred. However, othersources such as meat and the like may be used. Preferably the nucleobasesource is RNA.

Accordingly, the invention provides medicaments or nutritionalformulations comprising effective amounts of:

(a) glycine (component (a))

in association with one or more components selected from

(b) omega-3 PUFAs where desired in admixture with omega-6 PUFAs(component (b));

(c) L-arginine or other physiologically acceptable compound associatedwith the synthesis of nitric oxide, or mixtures thereof (component (c));and

(d) a nucleobase source (component (d)).

The dosage should be such that the medicaments or nutritionalformulations are effective for the prevention and/or treatment ofnephrotoxicity induced by macrolide immunosuppressive drugs.

One unit dose of such a medicament or nutritional formulation preferablycomprises 1.5 to 80 parts by weight of component (a) in association withthe following amounts of one or more components selected from (b) to(d): 0.1 to 20 parts by weight of component (b), 3 to 40 parts by weightof component (c) and 0.1 to 4.0 parts by weight of component (d).Particularly preferred one unit dose comprises 1.5 to 80 parts by weightof component (a) in association with the following amounts of one ormore components selected from (b) to (d): 2 to 5 parts by weight ofcomponent (b), 7.5 to 20 parts by weight of component (c) and 1.7 to 2.0parts by weight of component (d).

The amount of components (a) to (d) administered daily will convenientlycorrespond to 1.5 to 80 g for component (a), 0.1 to 20 g, preferably 2to 5 g, for component (b), 3 to 40 g, preferably 7.5 to 20 g, forcomponent (c) and 0.1 to 4.0 g, preferably 1.7 to 2.0 g, for component(d).

With respect to component (d) the above dosage is indicated for RNA,DNA, nucleosides or nucleotides. For nucleobases one weight unit ofnucleobases is regarded to be equivalent to 2.5 to 3.0 weight units ofRNA, DNA, nucleosides or nucleotides.

Where medicaments or nutritional formulations glycine in combinationwith one or more of the above-mentioned components (b), (c) and (d) areused, such medicaments or nutritional formulations will convenientlycomprise in one unit dose

(a) 1.5 to 80 parts by weight glycine,

in combination with one or more compounds selected from the groupconsisting of

(b) 2 to 5 parts by weight omega-3 polyunsaturated fatty acids;

(c) 7.5 to 20 parts by weight L-arginine, L-ornithine or glycerol, ormixtures thereof; and

(d) 1.7 to 2.0 parts by weight RNA.

Preferred medicaments or nutritional formulations comprise in one unitdose:

(a) from 1.5 to 80 parts by weight of glycine, in association with

(c) 3 to 40 parts by weight, preferably 7.5 to 20 parts by weight, ofarginine or an equivalent amount of one or more other physiologicallyacceptable compound associated with the synthesis of polyamines, or anequivalent amount of a mixture of arginine with such compound.

More preferably the medicaments or nutritional formulations of theinvention comprise component (a) in combination with component (c) at aweight ratio of 1:2 to 4:1, particularly preferred at a weight ratio of1:1 to 2:1.

Further preferred medicaments or nutritional formulations comprise inone unit dose:

(a) from 1.5 to 80 parts by weight of glycine, in association with

(b) 0.1 to 20 parts by weight, preferably 2 to 5 parts by weight, ofomega-3 PUFAs; and

(c) 3 to 40 parts by weight, preferably 7.5 to 20 parts by weight, ofarginine or an equivalent amount of one or more other physiologicallyacceptable compound associated with the synthesis of polyamines, or anequivalent amount of a mixture of arginine with such compound.

Omega-3 PUFAs are conveniently protected against peroxidation duringstorage of the formulation.

Physiologically acceptable ways of protecting omega-3 PUFAs againstperoxidation during storage are known in the art. They includephysiologically acceptable micro-encapsulation of omega-3 PUFAs and theuse of physiologically acceptable antioxidants in amounts sufficient toprotect the fatty acids.

A typical example suitable for use as physiologically acceptablemicro-encapsulation agents is starch. The micro-encapsulation can beeffected in a manner known per se. The micro-encapsules may be coated ina manner known per se, by physiologically acceptable coating agents suchas Gum Arabic.

Typical examples of antioxidants suitable for protecting fatty acidsagainst peroxidation include antioxidant vitamins such as Vitamin C,Vitamin E or mixtures thereof.

The amount of antioxidant added should be sufficient to preventperoxidation of the omega-3 PUFAs. Such amounts can be easilycalculated. In general, for convenience, any antioxydants employed toprevent peroxidation, will be employed in excess. It will be appreciatedthat the presence of any other agent administered in association withthe omega-3 PUFAs may require adjustment of the amount of antioxidant tobe employed.

The omega-3 PUFAs may be employed in a form suitable for thephysiological supply of omega-3 PUFAs, e.g. in free acid form, intriglyceride form, or in the form of physiologically acceptable naturalsources of omega-3 PUFAs. Such natural sources include linseed oil andfish oils such as menhaden oil, salmon oil, mackerel oil, tuna oil,cod-liver oil and anchovy oil. Said natural sources, in particular, thefish oils, comprise substantial amounts of omega-3 fatty acids. Wherethe omega-3 PUFAs are employed in triglyceride form, said triglyceridesmay comprise esters with other physiologically acceptable fatty acids.Preferred omega-3 PUFAs include eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), in free acid form, in triglyceride form orin form of natural sources having a high EPA and/or DHA content.

It will be appreciated that omega-3 PUFAs may be administered in higheramounts than those indicated hereinabove, and that such higher amountswill in general not impair the desired effect or provoke undesired sideeffects.

Compounds particularly suitable for use as component (c) in theformulation of the invention include L-arginine, glycerol andL-ornithine, most preferably L-arginine. Component (c) may be employedin free form, physiologically acceptable salt form, e.g. in the form ofa salt with phosphoric acid, citric acid, tartaric acid, fumaric acid,adipic acid or lactic acid, or in small peptide form. PreferablyL-arginine in free form is employed.

The term small peptides as used herein refers to peptides having from 2to 6, preferably from 2 to 4 amino acids.

As already indicated, omega-3 PUFAs will conveniently be administered inthe form of fish oils, protected or not against peroxidation. Such fishoils also comprises omega-6 PUFAs.

Omega-6 PUFAs have also a favourable effect on the immune response andon the resistance to infection upon surgery. Accordingly, diets of theinvention will conveniently further comprise omega-6 PUFAs.

For the purpose of the invention the omega-6 PUFAs may be in free acidform or in a form suitable for the physiological supply of omega-6PUFAs, e.g. in triglyceride form. Examples of omega-6 PUFAs particularlyappropriate for use according to the invention, include linoleic acidand arachidonic acid, linoleic acid being most preferred. Examples ofsuitable omega-6 PUFA sources are known in the art. They include fishoils and vegetable oils. Examples of omega-6 PUFA sources having a highlinoleic acid content such as safflower oil, sunflower oil, soya oil,cotton oil and corn oil.

Administration of a daily amount of omega-6 PUFAs in the range of from1.5 to 5.0 g will in general suffice to attain a favourable effect. Oneunit dose of the medicaments or nutritional formulation defined abovemay accordingly further contain 1.5 to 5 parts by weight of omega-6PUFAs.

In addition to components (b), (c) and (d), and omega-6 PUFAs furthercomponents may be added to the diets of the invention and may have abeneficial effect on the activity of glycine. An example of suchbeneficial components are omega-9 PUFAs. A preferred natural source forsuch fatty acid mixtures are fish oils. For taste and other reasons, thefish oils will, in oral application forms, preferably be used inencapsulated form.

Where the nutritional formulation of the invention is intended for useas a dietary supplement, the amount of energy supplied by it should notbe too excessive, in order not to unnecessarily suppress the patientsappetite. The supplement should conveniently comprise energy sources inan amount supplying from 150 to 1000 Kcal/day, preferably 250 to 500Kcal/day. The contribution of the nitrogen source, carbohydrate sourceand lipid source to the total daily caloric may vary within wide ranges.For use as a supplement, the administration may be in powder or liquidform.

The treatment period will coincide with the treatment with theimmunosuppressive drug.

The dietary supplement will conveniently be administered in the form ofunit doses suitable for administration of the supplement 1 to 4 timesper day. Where the nutritional formulations comprise energy sources, itis appropriate not to supply more than 1000 Kcal/day. Apart from thislimitation with respect to the energy supply, nutritional formulationsfor preventing and/or treating nephrotoxicity induced by macrolideimmunosuppressive drugs can and will conveniently be supplied in theform of formula diets or dietary supplements as described above.

Typical pharmacologically acceptable formulation forms (medicaments) fororal administration will further comprise pharmacologically acceptablediluents, carriers, vitamins, spices, pigments and/or other adjuvantswell known to the skilled person to be suitable for incorporation intosuch formulation and optionally a macrolide immunosuppressive drug.

The diets and formulations of the invention may be obtained in a mannerknown per se, e.g. by admixing the ingredients.

The invention is further illustrated by the following Examples which arenot intended in any way to limit the scope of the claimed invention.

EXAMPLE 1

Animal and Diets

Male Sprague-Dawley rats (Sasco) weighing approximately 200 g were fed asynthetic powdered diet containing 5% glycine and 15% casein (glycine)or 20% casein (control) for 3 days prior to initiation of cyclosporin Atreatment. Components of the powdered diet are shown in Table 1. Allanimals were given humane care in compliance with institutionalguidelines. After the prefeeding period, cyclosporin A (25 mg/kg solutedas 2.5 mg/ml in olive oil) or vehicle was given daily by oral gavage forapproximately 4 weeks. Four groups of 6-8 rats each (control, glycine,cyclosporin+control, cyclosporin+glycine) were studied. Animals weightand diet consumption was measured daily.

TABLE 1 Composition of Control and Glycine Diets Component ControlGlycine Casein 20.0 15.0 Glycine 0.0 5.0 Sucrose 50.0 50.0 Corn oil 5.05.0 Cellulose 5.0 5.0 Mineral mix 3.5 3.5 Vitamin mix 1.0 1.0DL-Methionine 0.3 0.3 Choline bitartrate 0.2 0.2 Corn starch 15.0 15.0

Glomerular Filtration Rate and Clinical Chemistry

Animals were placed in metabolic cages 48-72 hours prior to sacrifice,24 hour urine samples were collected and 0.5 ml of blood was drawn fromthe tail vein for blood creatinine measurements. On the day ofsacrifice, another 1 ml of blood was drawn from the vena cava andreplaced with an equal volume of Ringers lactate-solution. Serum urea(Coulombe, J. J. and L. Favreau. 1963. A new simple semimicro method forcolorimetric determination of urea. Clin. Chem. 9:102-108); andcreatinine (Heinegard, D. and G. Tiderstrom. 1973. Determination ofserum creatinine by a direct colorimetric method. Clin. Chim. Acta.43:305-310); as well as urine creatinine were measured using Sigma kitswhile serum alkaline phosphatase was measured enzymatically (bloodsamples were collected after approx. 4 weeks of cyclosporin Atreatment). Glomerular filtration rate was calculated from the ratio ofcreatinine in the urine/blood (Laiken, N. D. and D. D. Fanestil. 1985.Filtration and Blood Flow. In Physiological basis of medical practice.J. B. West, editor. Williams & Wilkins, Baltimore/London. 461-471),urine samples were collected after approx. 4 weeks of cyclosporin Atreatment.

Histology

On the day of sacrifice animals were anesthetized with pentobarbitol (50mg/lg), the abdomen was opened and the aorta was canulated with a 24French i.v. canula with the lip placed near the renal arterial branches.The aorta was clamped above and ligated beneath the renal branches andthe left kidney was rinsed with 5 ml normal saline and perfusion-fixedwith 4% paraformaldehyde solution in phosphate buffered saline. Thekidney was removed, cut in 0.5 thick slices and placed in 10% formaline.Hemtoxyllin-eosin sections were performed and analyzed microscopically.Dilated tubules were counted in 5 different low power fields (100×),necrotic cells and infiltrating macrophages were counted in 5 differenttubules at high power magnification (400×) and averages±SEM calculated.

Quantitation of Neutrophils

Blood smears were prepared from vena cava samples on the day ofsacrifice and stained with Wright Giemsa stain. Differential white bloodcell counts for 200 white blood cells were performed at high powermagnification (400×) and percentages calculated.

Statistics

For all statistics, one-way ANOVA and the Bonferonni post-hoc test wasused. p<0.05 was selected prior to the study to indicate significance.

RESULTS

Clinical Chemistry and Glomerular Filtration Rate

It is known that cyclosporin A elevates serum urea. Indeed, urea wasincreased over 70% in this study (Table 2). While glycine tended tominimize this increase, the effect was not statistically significant.Cyclosporin A also increased alkaline phosphatase (Table 2), and glycinetended to prevent the increase. Serum creatinine ranged from 0.4-0.6mg/dl in the control and glycine treated groups (FIG. 1) but was doubledsignificantly by cyclosporin A. This increase was prevented totally bythe glycine diet. Values are means±S.E.M. (P<0.01 by ANOVA; n=5-6 ineach group). a,p<0.05 compared to control; b,p<0.05 compared to thecyclosporin A group.

It is also known that cyclosporin A diminishes glomerular filtrationrate. In this study, values in the cyclosporin A group were about 30% ofthose of control or glycine alone groups (FIG. 2). This decrease inglomerular filtration rate was also totally prevented by glycine in thediet. A diet comprising 1% of the total caloric content as glycine wasalready sufficient to give this effect (results not shown). Values aremeans±S.E.M. (P<0.01 by ANOVA; n=5-6 in each group). a,p<0.05 comparedto control; b,p<0.05 compared to the cyclosporin A group. Significantchanges in urine volume were also noted (control, 9.1±1.6 ml/24 h;glycine, 6.7±0.8; cyclosporin A, 14.4±2.3; glycine+cyclosporin A,11.8±2.1).

TABLE 2 Effects of Cyclosporin A and a Glycine Diet on Serum Urea andAlkaline Phosphotase Levels Alkaline Urea Phosphotase (Mg/dl) (UL)Control 21.8 ± 1.8 46.5 ± 4.4 Glycine 20.6 ± 2.4 33.1 ± 5.6 CyclosporinA  34.9 ± 3.7^(a)  65.9 ± 12.6 Cyclosporin A + glycine  31.2 ± 0.6^(a)47.7 ± 8.8 Values are mean ± S.E.M.; a, p < 0.05 compared to controls byANOVA and Student-Newman-Keuls test (n = 5-6 in each group)

Histology and White Blood Cells

It is clear that cyclosporin A causes proximal tubular swelling andnecrosis with associated white blood cell infiltration as has beenreported previously.These effects were totally prevented by theglycine-containing diet. On average, proximate cell swelling andnecrosis as well as infiltrating macrophages were elevated significantlyby cyclosporin A (FIGS. 3, 4). Dilated proximal tubules (panel A of FIG.3) were counted in three different low power fields per slide(magnification 100×) and average±S.E.M. calculated. Necrotic cells(panel B of FIG. 3) were counted on five different tubules per slide athigh power (400×) (mean±S.E.M.*=p<0.05 by ANOVA). In FIG. 4 macrophageswere counted around 5 different tubules per slide at high magnification(400×) in hematoxyllin-eosin stained sections by a reviewer blinded totreatment and averages±S.E.M. calculated (*=p<0.05 by ANOVA). Further,there was a nearly 3-fold increase in neutrophils in blood smears fromcyclosporin A treated rats (FIG. 5). Blood smears were stained withWright Giemsa stain and differential cell counts were performed(*=p<0.05 by ANOVA).

EXAMPLE 2 Enteral Compositions

In the following compositions MM stands for “mineral mixture”, SM for“trace element mixture” and VM for “vitamin mixture”. The composition ofthese three mixtures is as follows:

Ingredients g/100 g MM Maltodextrins 34.40 K citrate/phosphate 34.60Magnesium dicitrate 8.20 Calcium chloride 8.00 Sodium citrate/chloride9.00 Citric acid 3.50 Choline tartrate 2.30 SM Maltodextrins 47.79Molybdenum-yeast 18.00 Chromium-yeast 9.20 Zinc sulfate 7.00Selenium-yeast 7.00 Ferrum(II) sulfate 6.92 Copper(II) gluconate 2.24Manganese(II) sulfate 1.12 Sodium fluoride 0.70 Potassium iodide 0.03 VMMaltodextrins 43.44 Sodium ascorbate 35.00 Vitamin E-Ac. 50% 16.00Niacinamide 1.55 Vitamin A-Acetate 1.20 Ca-D-Panthothenat 0.98 VitaminK₁ 1% 0.71 Vitamin B₁₂ 0.1% 0.30 Vitamin D₃ 0.28 Vitamin B₆ 0.20 VitaminB₁ 0.17 Vitamin B₂ 0.15 Folic acid 0.02 Biotin 0.01 Composition IComprising Glycine Water 77.40 Maltodextrins 12.28 Na/Ca caseinates 4.60Glycine 3.00 Lipids: Palm oil 2.33 Sunflower oil 0.26 Emulsifier NathinE 0.13 100.00 Composition II Comprising Glycine Water 77.40Maltodextrins 10.10 Na/Ca caseinates 4.60 Glycine 3.00 MM 2.00 SM 0.05VM 0.10 β-Carotine 0.03 Lipids: Palm oil 2.33 Sunflower oil 0.26Emulsifier Nathin E 0.13 100.00 Composition Comprising Glycine andArginine Water 77.40 Maltodextrins 8.93 Na/Ca caseinates 4.60 Glycine3.00 Arginine 1.17 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palmoil 2.36 Sunflower oil 0.23 Emulsifier Nathin E 0.13 100.00 CompositionComprising Glycine and Fish Oil (ω-3 fatty acids) Water 77.40Maltodextrins 10.10 Na/Ca caseinates 4.60 Glycine 3.00 MM 2.00 SM 0.05VM 0.10 β-Carotine 0.03 Lipids: Palm oil 1.32 Sunflower oil 0.23Emulsifier Nathin E 0.13 Fish Oil EPAX 3000 TG 1.04 100.00 CompositionComprising Glycine and RNA Water 77.40 Maltodextrins 9.96 Na/Cacaseinates 4.60 Glycine 3.00 Yeast extract RNA 0.14 MM 2.00 SM 0.05 VM0.10 β-Carotine 0.03 Palm oil 2.33 Sunflower oil 0.26 Emulsifier NathinE 0.13 100.00 Composition Comprising Glycine, Arginine and Fish Oil (ω-3fatty acids) Water 77.40 Maltodextrins 8.93 Na/Ca caseinates 4.60Glycine 3.00 Arginine 1.17 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03Lipids: Palm oil 1.32 Sunflower oil 0.23 Emulsifier Nathin E 0.13 FishOil EPAX 3000 TG 1.04 100.00 Composition Comprising Glycine, Arginineand RNA Water 77.40 Maltodextrins 8.79 Na/Ca caseinates 4.60 Glycine3.00 Arginine 1.17 Yeast extract RNA 0.14 MM 2.00 SM 0.05 VM 0.10β-Carotine 0.03 Lipids: Palm oil 2.33 Sunflower oil 0.26 EmulsifierNathin E 0.13 100.00 Composition Comprising Glycine, RNA and Fish Oil(ω-3 fatty acids) Water 77.40 Maltodextrins 9.96 Na/Ca caseinates 4.60Glycine 3.00 Yeast extract RNA 0.14 MM 2.00 SM 0.05 VM 0.10 β-Carotine0.03 Lipids: Palm oil 1.32 Sunflower oil 0.23 Emulsifier Nathin E 0.13Fish Oil EPAX 3000 TG 1.04 100.00 Composition Comprising Glycine,Arginine, RNA and Fish Oil (ω-3 fatty acids) Water 77.40 Maltodextrins8.79 Na/Ca caseinates 4.60 Glycine 3.00 Arginine 1.17 Yeast extract RNA0.14 MM 2.00 SM 0.05 VM 0.10 β-Carotine 0.03 Lipids: Palm oil 1.32Sunflower oil 0.23 Emulsifier Nathin E 0.13 Fish Oil EPAX 3000 TG 1.04100.00

As already set out above fish oil is a natural source for omega-3 PUFAswhereas sunflower oil is a natural source for omega-6 PUFAs.

What is claimed is:
 1. A method for the prophylactic and/or therapeutictreatment of renal dysfunction induced by cyclosporins or ascomycinscomprising enterally administering to a human being or other mammalsimultaneous with the treatment with a cyclosporin or ascomycin, amedicament or nutritional formulation comprising an amino acid componentconsisting essentially of glycine in an amount effective to inhibitand/or ameliorate the renal dysfunction, a carbohydrate source, andoptionally one or more components selected from vitamins, minerals,trace elements, and fibers.
 2. The method of claim 1 wherein thenutritional formulation is a dietary supplement.
 3. The method of claim2 wherein the dietary supplement consists essentially of glycine and acarbohydrate source.
 4. The method of claim 3 wherein the dietarysupplement further comprises a protein source.
 5. The method of claim 1wherein the nutritional formulation is a complete formula diet.
 6. Themethod of claim 1 wherein the medicament further comprises a cyclosporinor ascomycin.
 7. An enteral medicament or nutritional formulationcomprising a therapeutically effective amount of a cyclosporin orascomycin in combination with an amino acid component consistingessentially of glycine in an amount effective to inhibit and/orameliorate the renal dysfunction induced by said cyclosporin orascomycin, and 25 to 75% carbohydrate by weight of the medicament ornutritional formulation, and one or more components selected fromvitamins, minerals and trace elements, wherein glycine is present in anamount of about 0.2 to 60 g per 100 g dry weight of the medicament ornutritional formulation.
 8. The enteral medicament or nutritionalformulation of claim 7 which comprises one or more vitamins selectedfrom beta-carotene, Vitamin E, and Vitamin C.
 9. The enteral medicamentor nutritional formulation of claim 7 which comprises selenium.
 10. Theenteral medicament or nutritional formulation of claim 7 which comprisesfat and/or protein.
 11. The method of claim 2 wherein the dietarysupplement comprises 0.2 to 60 g glycine per 100 g dry weight and 25 to75% by weight carbohydrates.
 12. The method of claim 2 wherein thedietary supplement comprises 5 to 45 g glycine per 100 g dry weight and25 to 75% by weight carbohydrates.